We have developed and received patent protection for a biodegradable polyanhydride-based nanovaccine platform. Using F1-V as the immunogen, we have demonstrated its ability to induce, in a single administration, long-lived protective immunity in mice for up to 40 weeks against a lethal infection with Yersinia pestis, the causative agent of pneumonic plague. We propose to combine this nanovaccine platform with cyclic dinucleotide (CDN)-based innate immune inducers to design vaccines against multiple biodefense pathogens (i.e., Y. pestis and Bacillus anthracis), thereby breaking the one-bug, one-drug paradigm. Currently available vaccine formulations and adjuvants lack long-term stability, do not promote induction of antigen-specific cellular and humoral immunity, and/or are reactogenic. Our combination nanovaccine platform will overcome all these shortcomings, will be broadly applicable to infectious diseases, and will have application against non- defense respiratory pathogens as well. Furthermore, the nanovaccine is heat stable, thereby obviating the cold chain. Our central hypothesis, based on significant peer-reviewed preliminary data on the tunability and efficacy of our adjuvant platforms, is that polyanhydride nanovaccines together with CDNs can be used to formulate efficacious, single-dose vaccines against multiple biodefense pathogens. We will position this platform for preclinical studies that will advance the development of vaccine technologies specific for NIAID Category A priority agents by accomplishing the following Specific Aims, each of which is bounded by milestones, go/no-go decisions and fall-back positions: Aim 1. Optimize the immunization regimen of combination nanovaccine formulations to provide protection against lethal challenge with two biodefense agents. Aim 2. Evaluate the protective capabilities of the lead combination nanovaccine formulation from Aim 1 against lethal challenge with B. anthracis in rabbits and Y. pestis in non-human primates. Aim 3. Demonstrate enhanced storage shelf life performance of optimized lead combination nanovaccine formulation identified in Aims 1 and 2. Partners include: Iowa State University, who will refine the immunogenic dose and combination nanovaccine formulations, demonstrate the efficacy and protective capabilities of the vaccines in mice, and perform the storage shelf life studies; Aduro BioTech, who will prepare innate immune stimulators and evaluate interactions of combination nanovaccine formulations with human cells; and Lovelace Biomedical and Environmental Research Institute, who will determine the efficacy of the lead combination nanovaccine in rabbits and NHPs. At the end of the project period, we will deliver a single-dose combination nanovaccine formulation capable of safely inducing protective immunity against two biodefense pathogens that is ready for preclinical studies. The long-term impact of this work is protection of our militry troops prior to deployment as well as rapid immunization of an immunologically naive population following an exposure event.